GSM7011566: Mix_m6A-CT_scCUT&Tag; Mus musculus; OTHER
E14 mESCs were harvested, counted and centrifuged for 5 min at 350×g at room temperature. Aliquots of cells (~1,000,000 cells) were resuspended in 250 μL Nuclei Extraction Buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.020 % Triton X-100) and incubated on ice for 1 min. The mixture was diluted with 1 mL Nuclei Dilution Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 0.1 U/μL RiboLock RNase inhibitor) and centrifuged for 5 min at 600×g at 4 °C. Nuclei were washed once with 1 mL Nuclei Dilution Buffer and counted. E14 EPSC and MEF nuclei were similarly isolated with the following conditions: 1 min on ice 0.015 % Triton X-100 for EPSC; 5 min on ice in 0.2 % NP-40 for MEF. Aliquots of nuclei (150,000 nuclei in total per assay) were incubated with 1 μg primary antibody (rabbit IgG or rabbit anti-m6A: MilliporeSigma, #ABE572) in 50 μL Antibody Binding Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) for 2 hours on rotator at 4 °C. Afterwards, nuclei were incubated with 1 μg secondary antibody (guinea pig anti-rabbit: Novus Biologicals, #NBP1-72763) in 50 uL Antibody Binding Buffer for 30 min on the nutator at room temperature. Nuclei were then centrifuged for 5 min at 600×g at room temperature and two rounds of washes with 200 μL Wash Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 0.1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) were performed. The nuclei were resuspended in 50 μL high-salt Binding Buffer (20 mM HEPES pH7.5, 300 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) with 2.5 μL pAG-Tn5 (EpiCypher, #15-1117) and incubated for 1 hour on the nutator at room temperature. The nuclei were then washed twice with high-salt Wash Buffer (20 mM HEPES pH7.5, 300 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 0.1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) and resuspended in 50 μL Reverse Transcription & Tagmentation Buffer (1X SuperScript IV buffer along with, 1 M Betaine, 5 mM DTT, 2 μM Random Hexmer Primers, 0.5 mM each dNTP mix, 1X Roche Complete Protease Inhibitor Cocktail, 1 U/μL RiboLock RNase inhibitor, 4 U/μL SuperScript IV) for 2 hours at 37 °C. When reaction was done, the nuclei were resuspended with 50 μL cold Antibody Binding Buffer and placed on ice. All steps after nuclei extraction were performed in 0.2-ml tubes to minimize nuclei loss. The nuclei were counted and diluted to a concentration based on the targeted nuclei number. Seven microliters of 10x Genomics ATAC Buffer B was added to 8 μl of nuclei, pipetting several times to mix. Most of the remaining steps were performed according to the Chromium Next GEM Single Cell Multiome protocol Rev E (10x Genomics), skipping the Step 1 'Transposition' and continuing from Step 2.0 'Generation & barcoding'. A qPCR cycle check was performed after pre-amplification step using 1:100 amplicon. Indexed CT libraries were prepared with the following PCR program: 45 sec at 98 °C, followed by 23 cycles (ESC IgG-CT) or 15 cycles (ESC and mixed cell-lines m6A-CT) of 15 sec at 98 °C and 10 sec at 60 °C, with a final extension at 1 min at 72 °C. For cDNA libraries, cycle numbers used in cDNA amplification and sample index PCR were 7 cycles and 12 cycles, respectively. The final cDNA libraries and CT libraries were sequenced using an Illumina NovaSeq 6000, PE150 and PE50 system, respectively. Library was performed according to the manufacturer's instructions (single cell Multiome Rev E protocol, 10x Genomics).